Chromosome instability (CIN) is an important component in several human health problems including cancer, birth defects, and infertility. The Gorbsky lab discovered a new source of CIN that was termed cohesion fatigue. Cohesion fatigue is the progressive, asynchronous separation of sister chromatids in cells delayed at metaphase. The overall goals of this project are to map the downstream consequences of cohesion fatigue, the mechanisms by which chromatids surrender cohesion, and the upstream pathways that modulate the cell sensitivity to cohesion fatigue. In Aim 1, advanced microscopy at both the single cell level and population level will be used to track the chromosome abnormalities that arise from cohesion fatigue. Cohesion fatigue has the potential to simultaneously generate the two types of gross chromosome aberrations that often arise during oncogenesis, changes in whole chromosome number (aneuploidy) and large segmental chromosome duplications, deletions, and translocations. In addition, cohesion fatigue is highly likely to lead to the formation of micronuclei, which have been implicated as sites of massive DNA damage. Aim 2 will determine the mechanisms of cohesin release during cohesion fatigue through experiments that lock individual joints of the cohesin protein complex. In addition, quantitative mass spectrometry will be used to analyze cohesin components that are removed or altered in their post-translational modifications during cohesion fatigue. Aim 3 will map the upstream pathways that regulate sensitivity to cohesion fatigue, concentrating on defects in transformed cells that may exacerbate CIN. Transformed cells often exhibit defects in cell cycle regulators, and cohesion genes are among the most often mutated in human tumors. Thus, transformed cells may be highly susceptible to cohesion fatigue. This aim will test how alterations of cell cycle regulators induce metaphase delays and how these delays synergize with transformation-associated defects in spindle microtubule dynamics and chromosome cohesion to promote cohesion fatigue. Aim 4 extends the analysis of cohesion fatigue to budding yeast to examine the conservation of cohesion fatigue regulators in mitosis and to test specific hypotheses about how cohesion fatigue contributes to premature loss of cohesion between homologous chromosomes during meiosis. Recent evidence implicates decay in chromosome cohesion as a contributor to the maternal age effect, whereby the oocytes of older women show a greatly increased incidence of aneuploidy. In mammalian gametes, cohesion fatigue may be an important causative factor in meiotic aneuploidy, contributing to birth defects and infertility.